We intend to develop new chemical classes of antibacterials by focusing on an under-exploited target in a well-validated pathway. Specifically, we will develop and apply a high throughput screen for inhibitors of the Staphylococcus aureus replicative DNA helicase (RDH), an essential target in the DNA replication pathway. Our overall goal is to identify specific bacterial helicase inhibitors and develop them into novel antibiotics to treat resistant organisms. In Phase I, we will clone, over-express, and purify the S. aureus RDH and any necessary ancillary proteins. We will then develop a high-throughput screen (HTS) and apply it to a diverse library of over 100,000 discrete small molecule compounds and purified natural products. The HTS assay will measure the time-resolved fluorescence signal following the helicase catalyzed unwinding of a fluorescent oligo annealed to a quencher oligo. This assay will detect inhibitors of any of the multiple essential helicase functions, including strand unwinding, ATPase-coupled translocation, DNA binding, and protein-protein interactions. We will confirm hits and eliminate non-specific DNA-binding compounds by rapid assays for inhibition of a second replication function (DNA polymerase IIIC). Promising compounds from this screen will be tested for ability to inhibit the growth and viability of S. aureus and a variety of Gram(+) and Gram(-) bacterial pathogens. Specificity for DNA replication and cytotoxicity to mammalian cells in culture will also be evaluated to generate a series of validated hits. The specific aims are to (1) develop a high-throughput screening assay for the essential RDH of S. aureus, (2) screen a diverse compound library to identify and confirm inhibitors of S. aureus RDH; and (3) prioritize confirmed screening hits for spectrum, potency, mechanism, and selectivity. In Phase II, we will characterize the mechanism of action of the validated hits in more detail and optimize the most promising of these structures utilizing a rational drug design approach to develop antibacterial lead compounds. [unreadable] [unreadable]